Humanized anti-CD4 antibody with immunosuppressive properties

ABSTRACT

A humanized antibody derived from mouse monoclonal anti-CD4 antibody B-F5 is able to activate CD25+CD4+ regulatory T cells and is useful for preparing immunosuppressive compositions.

This application is a continuation under 35 U.S.C. §120 to U.S. patentapplication Ser. No. 11/217,402, filed Sep. 2, 2005, now U.S. Pat. No.7,452,981 which was a continuation under 35 U.S.C. §120 to Internationalapplication number PCT/EP2004/002888, filed 19 Mar. 2004, which claimspriority under 35 U.S.C. §119 to European application no. 03.290725.5,filed 21 Mar. 2003, and European application no. 03.290942.6, filed 16Apr. 2003, the entireties of which are incorporated by reference herein.The Sequence Listing filed electronically herewith is also herebyincorporated by reference in its entirety (File Name: 060-002_Seq_List;File Size: 6 KB; Date Created Oct. 7, 2008).

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention relates to a humanized anti-CD4 antibody, and to its usefor immunomodulation.

2. Brief Description of the Related Art

Autoimmune diseases as well as graft rejection result from aninappropriate immune response to tissue antigens: self antigens in thefirst case, and allograft antigens in the second one.

Autoimmune diseases include for instance rheumatoid arthritis, type Idiabetes, multiple sclerosis, Crohn's disease, ulcerative colitis,atopic dermatitis, etc.

Conventional treatments for these immunological disorders involveimmunosuppressive drugs. However these drugs induce a generalimmunosuppression, resulting in inhibition of not only the harmfulfunctions of the immune system, but also the useful ones. As aconsequence, they induce side effects, such as opportunistic infections.

As an alternative approach, it has been proposed to useimmunosuppressive monoclonal antibodies (mAbs) against cell-surfacemolecules, in order to remove specific lymphocyte subsets (depletingantibodies) or to inhibit the function of a target surface moleculewithout killing the cell bearing it (nondepleting-antibodies).

It is generally agreed that CD4+ T cells play a major part in initiatingand maintaining autoimmunity. Accordingly, it has been proposed to usemAbs against CD4+ T cells surface molecules, and in particular anti-CD4mAbs, as immunosuppressive agents. Although numerous clinical studiesconfirmed the potential interest of this approach, they also raisedseveral issues to be addressed in order to make anti-CD4 mAbs moresuitable for use in routine clinical practice.

By way of example, B-F5 antibody (murine IgG1 anti-human CD4) was testedin different autoimmune diseases:

in rheumatoid arthritis patients, several open studies suggested apositive clinical effect of B-F5 at a daily dose of at least 20 mg(Racadot et al. Clin. Exp. Rheumatol. 10 (4): 365-74; 1992; Wendling etal., Clin. Rheumatol., 11 (4): 542-7, 1992). However, the resultsobserved in a placebo controlled trial with a daily dose of 20 mg for 10days did not show a significant improvement (Wendling et al. J.Rheumatol.; 25 (8): 1457-61, 1998).

in psoriasis, an improvement in psoriatic lesions was observed followinga treatment at a dose of 0.2 mg/kg/day to 0.8 mg/kg/day for 7 or 8 days(Morel et al. J. Autoimmun., 5 (4): 465-77, 1992);

in multiple sclerosis (MS) patients, some positive effects were observedafter a 10 days treatment in patients with relapsing-remitting forms,some of who were relapse-free at the 6th month post-therapy (Racadot etal., J. Autoimmun., 6 (6):771-86, 1993); similar effects were observedby Rumbach et al. (MultScler; 1 (4): 207-12, 1996);

in severe Crohn's disease, no significant improvement was observed inpatients receiving B-F5 at a dose of 0.5 mg/day/kg for 7 consecutivedays or of 0.5 mg/day/kg on the first day (day 0) and of 1 mg/day/kg ondays 1-6 (Canva-Delcambre et al., Aliment Pharmacol. Ther. (5):721-7,1996);

in prevention of allograft rejection, a modification of the biologicalparameters, indicating an action of B-F5 in vivo at a 30 mg/daily dosewas reported. However, it was reported that B-F5 bioavailability was notsufficient to allow its use for prophylaxis of allograft rejection(Dantal et al. Transplantation, 27; 62(10):1502-6, 1996).

It appears from the above that a first issue to be solved is the need ofusing high doses of mAb to obtain a clinical improvement. This mayresult inter alia from the poor accessibility to the mAb of thelymphocytes in the target tissues. The use of higher doses may result inan excessive action on blood lymphocytes, inducing unwanted sideeffects.

Another drawback of therapy with monoclonal antibodies in humans is thatthese antibodies are generally obtained from mouse cells, and provokeantimouse responses in the human recipients. This not only results in alesser efficiency of the treatment and even more of any future treatmentwith mouse monoclonal antibodies, but also in an increased risk ofanaphylaxis.

This drawback can, in principle, be avoided by the use of humanizedantibodies, obtained by grafting the complementarity-determining regions(CDRs) of a mouse monoclonal antibody, which determine theantigen-binding specificity, onto the framework regions (FRs) of a humanimmunoglobulin molecule. The aim of humanization is to obtain arecombinant antibody having the same antigen-binding properties as themouse monoclonal antibody from which the CDR sequences were derived, andfar less immunogenic in humans.

In some cases, substituting CDRs from the mouse antibody for the humanCDRs in human frameworks is sufficient to transfer the antigen-bindingproperties (including not only the specificity, but also the affinityfor antigen). However, in many antibodies, some FR residues areimportant for antigen binding, because they directly contact the antigenin the antibody-antigen complex, or because they influence theconformation of CDRs and thus their antigen binding performance.

Thus, in most cases it is also necessary to substitute one or severalframework residues from the mouse antibody for the human correspondingFR residues. Since the number of substituted residues must be as smallas possible in order to prevent anti-mouse reactions, the issue is todetermine which amino acid residue (s) are critical for retaining theantigen-binding properties. Various methods have been proposed forpredicting the more appropriate sites for substitution. Although theyprovide general principles that may be of some help in the first stepsof humanization, the final result varies from an antibody to another.Thus, for a given antibody, it is very difficult to foretell whichsubstitutions will provide the desired result.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a DNA sequence encoding mouse B-F5 V_(H) region (SEQ IDNo. 5).

FIG. 2 depicts a DNA sequence encoding mouse B-F5 V_(K) region (SEQ IDNo. 6).

FIG. 3 shows an alignment of the polypeptide sequences of B-F5 (SEQ IDNo. 10), FK-001 (SEQ ID Nos. 11, 12, 13, and 14, respectively, from leftto right), L4L, and L4M (SEQ ID No. 2).

FIG. 4 shows an alignment of the polypeptide sequences of B-F5 (SEQ IDNo. 9, M26 (SEQ ID Nos. 15, 16, 17, and 18, respectively, from left toright), H37L, and H37V (SEQ ID No. 1).

FIG. 5 depicts the fragment of the plasmid encoding the VH region ofhumanized BF-5; the complete nucleic acid sequence is SEQ ID No. 7, theunderlined nucleic acid sequence is SEQ ID No. 3, the amino acidsequence is SEQ ID No. 1).

FIG. 6 depicts the fragment of the plasmid encoding the VK region ofhumanized BF-5; the complete nucleic acid sequence is SEQ ID No. 4, theunderlined nucleic acid sequence is SEQ ID No. 4, the amino acidsequence is SEQ ID No. 2).

FIG. 7 shows the results of the ELISA assays, wherein murine and hB-F5scould moderately inhibit ConA-induced proliferation, but the activitiesvaried from antibody to antibody and/or from donor to donor.

FIG. 8 shows the results of the ELISA assays, wherein murine and hB-F5swere able to inhibit Ag-specific PBMC proliferation induced by PPD.

FIG. 9 shows a test of suppressive activity, wherein the negativecontrol (no activation) is preCD25; 0.5 μg/ml OKT-3 (positive control,full activation) is preCD25-CD3; 5 μg/ml hB-F5 (Test 1) is preCD25-CD4;30 μg/ml hB-F5 (Test 2) is preCD25-CD4.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have however attempted the humanization of mouse B-F5, andhave succeeded in producing humanized B-F5 (hereinafter referred to ashB-F5) having the same CD4 binding properties than parent mouse B-F5.

Furthermore, they have found that, surprisingly, hB-F5 has an in vivooptimal immunosuppressive effect at far lower doses than thosepreviously used with parent B-F5, and than those currently used withother anti-CD4 monoclonal antibodies.

Actually, the inventors have observed that hB-F5 provided an effectiveimmunosuppression, reflected by a positive clinical effect in rheumatoidarthritis patients, when used in a 10 days treatment at a dose as low as1 mg/day, and preferably at a dose of 5 mg every second day.

The present invention provides a humanized antibody (hB-F5) derived frommouse B-F5 MAb, wherein said hB-F5 antibody has V domains defined by thefollowing polypeptide sequences:

H chain V domain: (SEQ ID NO: 1)EEQLVESGGGLVKPGGSLRLSCAASGFSFSDCRMYWLRQAPGKGLEWIGVISVKSENYGANYAESVRGRFTISRDDSKNTVYLQMNSLKTEDTAVYYCSASYYRYDVGAWFAYWGQGTLVTVSS L chain V domain: (SEQ ID NO: 2)DIVMTQSPDSLAVSLGERATINCRASKSVSTSGYSYIYWYQQKPGQPPKLLIYLASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQHSRELPW TFGQGTKVEIK.

Generally, a hB-F5 antibody of the invention further comprises a humanconstant region (Fc). This constant region can be selected amongconstant domains from any class of immunoglobulins, including IgM, IgG,IgD, IgA and IgE, and any isotype, including IgG1, IgG2, IgG3 and IgG4.Preferred constant regions are selected among constant domains of IgG,in particular IgG1.

The present invention also includes any fragment of an hB-F5 antibodycomprising the V regions thereof. This comprises in particular Fab,Fab′, F(ab)′2, Fv and scFv fragments.

The invention also encompasses a polynucleotide selected among:

a polynucleotide encoding a polypeptide of SEQ ID NO: 1

a polynucleotide encoding a polypeptide of SEQ ID NO: 2.

Preferably, said polynucleotide is selected among:

a polynucleotide of SEQ ID NO: 3;

a polynucleotide of SEQ ID NO: 4.

Polynucleotides of the invention can easily be obtained by thewell-known methods of recombinant DNA technology and/or of chemical DNAsynthesis.

A polynucleotide encoding the V domain of the H chain or of the L chainof a hB-F5 antibody may be fused with a polynucleotide coding for theconstant region of a human H or L chain, for the purpose of expressingthe complete H and L chains obtained in this way; a sequence coding asignal peptide allowing the secretion of the protein can also be added.These recombinant polynucleotides are also part of the invention.

The invention also provides expression cassettes wherein apolynucleotide of the invention is linked to appropriate controlsequences allowing the regulation of its transcription and translationin a chosen host cell, and recombinant vectors comprising apolynucleotide or an expression cassette of the invention.

These recombinant DNA constructs can be obtained and introduced in hostcells by the well-known techniques of recombinant DNA and geneticengineering.

The invention also comprises a host cell, transformed by apolynucleotide of the invention.

Useful host-cells within the framework of the present invention can beprokaryotic or eukaryotic cells. Among suitable eukaryotic cells, onewill mention, by way of example, plant cells, cells of yeasts such asSaccharomyces, cells of insects such as Drosophila, or Spodoptera, andmammal cells such as HeLa, CHO, 3T3, C127, BHK, COS, etc. . . . .

The construction of expression vectors of the invention, and thetransformation of host-cells can be made by the standard techniques ofmolecular biology.

An hB-F5 antibody of the invention can be obtained by culturing a hostcell containing an expression vector comprising a nucleic acid sequenceencoding said antibody, under conditions suitable for the expressionthereof, and recovering said antibody from the host cell culture.

The present invention also comprises a therapeutic compositioncomprising a hB-F5 antibody of the invention or a fragment thereof, asdefined above.

Preferably, said composition is a composition for parenteraladministration, formulated to allow the administration of a dose of from0.1 to 10 mg, advantageously of from 1 to 5 mg of hB-F5.

More specifically, the invention encompasses the use of an hB-F5antibody of the invention or a fragment thereof, for preparing animmunosuppressive composition. Said immunosuppressive composition isuseful in particular for the treatment or prevention of diseases such asgraft rejection, graft-versus-host reaction or host-versus-graftreaction, or autoimmune diseases including for instance myocarditis,diabetes mellitus, psoriasis, lupus erythematosus, Crohn's disease,multiple sclerosis, rheumatoid arthritis, etc.

Moreover, the inventors have found out that hB-F5 was able to activate aparticular subset of T CD4+ cells, namely CD4+CD25+ cells.

CD25+CD4+ regulatory T cells (Treg cells) constitute 5-10% of peripheralCD4+ T cells. They were first described in 1995 by Sakaguchi et al. (J.Immunol., 155: 1151-1164) as regulatory cells in mice. When activated,these cells are able to suppress both CD4+ and CD8+ T cell activationand proliferation. Later, CD25+CD4+ suppressor T cells have also beenfound in humans (Jonuleit et al., J. Exp. Med. 193, 1285-1294, 2001;Levings et al., J. Exp. Med. 193, 1295-1302, 2001; Dieckmann et al., J.Exp. Med. 193, 1303-1310 2001). Numerous articles have been publisheddescribing the immunosuppressive role of these cells in differentautoimmune disease models and in vitro systems (for review, see forinstance Shevach, J. Exp. Med., 193, 11, 41-46, 2001). Ex vivo activatedCD4+CD25+ Treg cells have also been shown to be effective at preventinggraft-versus-host disease (Taylor et al., Blood, 99, 3493-3499, 2002;Cohen et al., J. Exp. Med. 196, 401-406, 2002; Hoffmann et al., J. Exp.Med. 196,389-399, 2002). Thus, providing means for activating CD4+CD25+Treg cells is of great interest.

The invention also relates to the use of the hB-F5 antibody of theinvention, or of the parent antibody B-F5, to activate in vitroCD25+CD4+ regulatory T cells.

Preferably, the hB-F5 antibody of the invention is added to theCD25+CD4+ regulatory T cells at a concentration 1 μg/ml from 10 μg/ml.

EXAMPLES

The present invention will be further illustrated by the followingadditional description, which refers to examples illustrating theproperties of hB-F5 antibodies of the invention. It should be understoodhowever that these examples are given only by way of illustration of theinvention and do not constitute in any way a limitation thereof.

Example 1 Construction of Humanized B-F5

Design of Humanized B-F5 VH and VK Regions

DNA sequences encoding mouse B-F5 V_(H) and V_(K) regions arerespectively shown in FIG. 1 and FIG. 2 and under sequence identifiersSEQ ID NO: 5 and SEQ ID NO: 6. The human V_(H) and V_(K) on which themouse CDRs are grafted were selected by searching databases for human VHmost like the original mouse B-F5 V_(H) and V_(K). V_(H) region of ahuman antibody (M26; Accession Number A36006) had the highest homologywith B-F5 V_(H). V_(K) region of another human antibody (FK-001;NAKATANI et al., Biotechnology, 7 (1989), 805-810)) had the highesthomology with B-F5 V_(K).

Two types of V_(K) differing between them in that the 4th residue wasLeucine or Methionine were constructed and designated as L4L and L4M.Two types of VH differing between them in that the 37th amino acidresidue was Leucine or Valine, were constructed and designated as H37Land H37V. The alignment of the polypeptide sequences of B-F5, FK-001,L4L, and L4M is shown in FIG. 3. The alignment of the polypeptidesequences of B-F5, M26, H37L, and H37V is shown in FIG. 4. The FRresidues previously reported to be important for the packing of CDRs(Chothia et al., Nature, 342 (1989), 877; Foote et al., J. Mol. Biol.,224 (1992), 487) are boxed.

By combining these VH and VK, 4 versions of V regions were designed.

Expression of Humanized B-F5

The subsequent steps for production of humanized B-F5 were the same asthose disclosed in U.S. Pat. No. 5,886,152 for humanized B-B10.

Briefly, expression plasmids for the H chain (VH humanized region fusedto the constant region of a humany-1 chain (TAKAHASHI et al., Cell, 29(1982), 671-679)) and the L chain (VK humanized region fused to theconstant region of FK-001K chain) of humanized B-F5 were constructedseparately. In these plasmids, the expression of humanized B-F5 isdriven by the promoter/enhancer of the gene of human monoclonal IgM,FK-001. FIGS. 5 and 6 respectively show the fragments of the plasmidsencoding the VH and VK regions of humanized BF-5. The sequences encodingthe V region are underlined and the corresponding polypeptide sequencesare indicated above the nucleotide sequence. Both plasmids and pSV2neowere simultaneously introduced into mouse myeloma Sp2/0 (ATCC CRL-1581)using Lipofectin. Transfectomas producing human IgG were selected byELISA, using an anti-human IgG (y chain) antibody and an anti-human Ig Kchain antibody.

Example 2 Characterisation of the Different Versions of Humanized B-F5Estimation of CD4 Binding Activity

Culture supernatants of transfectomas producing the four versions ofhB-F5 were collected, and concentrated. The different antibodies werepurified from culture supernatants by affinity chromatography usingprotein A Sepharose and assessed for their CD4 binding activity bymeasuring, by means of competitive ELISA, their inhibitory activitiesagainst the binding of biotinylated mB-F5 to soluble CD4 coated onmicrotiter plates. Incubation time is 2 hours for 37 C and overnight for4 C.

The relative binding activities of hB-F5s (binding activity of mB-F5 wastaken as 100%) are shown in Table I below

TABLE I Relative binding activity Antibody Temp (° C.) (% of mB-F5)H37L/L4L 4 80 37 30 H37L/L4M 4 80 37 30 H37V/L4L 4 10-20 37 10 H37V/L4M4 10-20 37 10

From the results shown in Table I, it appears that the 37th residue ofV_(H), Leucine, is critical to maintain CD4 binding activity of hB-F5because the CD4 binding activity is several-fold reduced by conversionof ³⁷Leu to ³⁷Val. On the contrary, the 4^(th) residue of V□ is found tobe not so important for the CD4 binding activity. As the structuraldifference between ³⁷Leu and ³⁷Val of V_(H) is not clearly demonstratedby molecular modeling, the superiority of H37L to H37V in CD4 bindingactivity was unexpected.

H37L/L4L and H37L/L4M were chosen for evaluating the in vitro biologicalactivities.

Investigation of the In Vitro Biological Activities of Humanized B-F5

The in vitro biological activities of mouse B-F5 and humanized B-F5s(H37L/L4M IgG1 and H37L/L4L IgG 1) were evaluated. Humanized B-F5s ofIgG2 type (H37L/L4M IgG2 and H37L/L4L IgG2) were also tested.

The in vitro biological activities of mB-F5 and the four types of hB-F5swere evaluated using peripheral blood mononuclear cells (PBMCs) fromhealthy donors. PBMCs were activated by ConA (2.5 μg/ml, 3 days) of PPD(10 μg/ml, 4 days) in the presence of murine or hB-F5s, and weremonitored for their proliferative responses by ³H-thymidineincorporation.

The results are shown in FIGS. 7 and 8. Murine and hB-F5s couldmoderately inhibit ConA-induced proliferation, but the activities variedfrom antibody to antibody and/or from donor to donor (FIG. 7). Also,murine and hB-F5s were able to inhibit Ag-specific PBMC proliferationinduced by PPD (FIG. 8).

IgG1 type of hB-F5 inhibited PPD-induced proliferation more effectively(as high as 70% inhibition, FIGS. 7 and 8) than Mb-F5. IgG1 type seemedto be more effective than IgG2 type of which inhibitory activity wasalmost the same as mB-F5. For IgG2 type of H37L/L4M and H37L/L4Linhibitory activities of B-F5s against PPD-induced PMBC proliferationwere as follows: H37L/L4M IgG1>H37L/L4L IgG1>H37L/L4M IgG2=H37L/L4LIgG2=mB-F5.

Considering the efficacy of the in vitro biological activity and thesmaller number of mouse amino acids, H37L/L4M IgG1 was chosen forfurther evaluation.

Example 3 Preliminary Evaluation of the Effect of hB-F5 on Patients withRheumatoid Arthritis (RA)

The effect of hB-F5 (H37L/L4M IgG1) was tested on RA patients.

The conditions of the assay are as follows:

Each patient received a 10 days treatment consisting of 5 injections of5 mg of hB-F5 (an injection every 2nd day).

The results for 3 different patients are shown in Tables II-IV below:

Patient 1 (Table II):

Diagnosis: Rheumatoid Arthritis, Activity 2

Rheumatoid factor: 2; Stage: 2

Sex: F; Age: 65; Onset of the disease: 1965

Additional therapy: Diclophenac 150 mg/day

TABLE II After treatment Before During treatment (days) (weeks) ClinicalInvestigations Treatment 2 4 6 8 10 4 Estimation of pain in joints 4.5 22 1.5 3 2.2 3.5 (0-10) Morning stiffness in minutes 360 0 0 90 90 120 20Severity of Physician 3 3 3 2.5 3 3 3 condition (1-5) Patient 3 3 3 3 33 3 Number of swollen joints 6 6 4 3 2 2 7 Number of painful joints 2512 6 7 13 13 23 Swelling index (0-30) 8 6 4 2 3 9 Power in hand Right 1715 20 22 12 20 15 Left 10 10 10 15 12 19 12 Estimation of tiredness(0-10) 7.7 4 2.3 2 2.3 3.1 3 Estimation of Patent 3 3 4 3 5 2 treatmentPhysician 3 3 4 3 5 2 effects Erythrocyte sedimentation 35 34 25 rateC-Reactive Protein 4.0 2 2.5

Patient 2 (Table III):

Diagnosis: Rheumatoid Arthritis, Activity 3

Rheumatoid factor: 2; Stage: 2

Sex: F; age: 48 Onset of the disease: 2000

Additional therapy. Diclophenac 150 mg/day

TABLE III After treatment Before During treatment (days) (weeks)Clinical Investigations Treatment 2 4 6 8 10 4 Estimation of pain injoints 8.2 8.2 5 2.9 2.2 0.6 (0-10) Morning stiffness in minutes 240 120120 60 20 10 Severity of Physician 4 3 3 3 3 2 condition (1-5) Patient 44 3 3 3 2 Number of swollen joints 13 12 11 11 5 5 Number of painfuljoints 22 22 16 15 13 7 Swelling index (0-30) 15 14 12 11 5 5 Power inhand Right 30 30 28 34 36 40 Left 22 20 18 18 22 28 Estimation oftiredness (0-10) 8.7 5.1 2.2 2.2 1.1 0.7 Estimation of Patent 3 4 4 4/55 treatment Physician 3 2 3 4/5 5 effects Erythrocyte sedimentation 3538 35 rate C-Reactive Protein 1.2 0.2 0.8

Patient 3 (Table IV):

Diagnosis: Rheumatoid Arthritis, Activity 3

Rheumatoid factor: 3; Stage: 2

Sex: F; Age: 49; Onset of the disease: 1989

Additional therapy. Diclophenac 150 mg/day

TABLE IV After treatment Before During treatment (days) (weeks) ClinicalInvestigations Treatment 2 4 6 8 10 1 2 Estimation of pain in joints 7.97.6 7.6 7.2 5.0 3.0 1.5 1.3 (0-10) Morning stiffness in 360 0 0 0 0 0 0minutes Severity of Physician 4 3 3 3 3 3 2 2 condition (1-5) Patient 54 4 3 3 2 2 Number of swollen joints 10 7 7 6 5 5 5 5 Number of painfuljoints 30 24 24 15 11 11 10 9 Swelling index (0-30) 15 12 12 9 7 7 6Power in hand Right 24 30 30 36 48 48 50 50 Left 24 30 30 38 40 34 40 42Estimation of tiredness (0-10) 8.5 7.2 5.2 0 0 0 0 0 Estimation ofPatent 3 3 3 5 5 5 treatment Physician 3 4/3 4 4 5 5 5 effectsErythrocyte sedimentation 61 53 42 45 41 rate C-Reactive Protein 8 3.73.3

Example 4 Activation of CD4+CD25+ Treg Cells by hB-F5

Isolation of T Cells:

1) T Regulatory Cells (Tregs):

CD25+ cells are isolated using CD25 microbeads;

Depletion of contaminations: CD14−, CD8−, CD19− positive cells is madewith CD14/CD8/CD19DYNALbeads;

Depletion of CD45RA+ cells is made with CD45RA mAb+anti-mouseDYNALbeads: purity: >95% CD4+CD25+ Tregs

2) Effector Cells

CD4+ T cells are isolated using CD4 microbeads

Depletion of CD45RO+ cells is made with CD45RO+mAb+anti-mouseDYNALbeads; purity: >98% CD4/CD45RA+, CD25− effector T cells

3) Test System:

CD25+ Tregs from donor A are cocultured for 2 days with syngenicCD2-depleted PBMC, without additions (negative control=no activation=nosuppressive activity), or in the presence of 0.5 μg/ml anti-CD3(OKT-3=positive control=full activation of Tregs), or in the presence of5 μg/ml or 30 μg/ml hB-F5.

After extensive washing of pre-cultured cells, CD25+ Tregs cells areisolated and treated by y-radiation (3000 rad).

4) Test of Suppressive Activity:

Pre-cultured CD25+ Tregs cells are cocultured for 4 days with freshlyisolated CD4+ effector T cells (1:1) from donor B in the presence ofAPC's (CD2-depleted PBMC) from donor A (syngenic for pre-cultured Tcells (no additional activation), allogeneic for effector T cells(=allogeneic mixed lymphocyte reaction). Then, cells are incubated for16 h with 3H Thymidine, and proliferation of effector T cells isdetected.

The results are shown in FIG. 9.

Legend of FIG. 9:

negative control (no activation)=preCD25;

0.5 μg/ml OKT-3 (positive control, full activation)=preCD25-CD3;

5 μg/ml hB-F5 (Test-1)=preCD25-CD4;

30 μg/ml hB-F5 (Test-2)=preCD25-CD4.

What is claimed is:
 1. A humanized anti-CD4 antibody, wherein the humanized anti-CD4 antibody comprises a sequence comprising the complementarity-determining regions (CDRs) of SEQ ID NOs: 10 and 9, displayed in FIGS. 3 and 4, and wherein the humanized anti-CD4 antibody is capable of activating CD4⁺CD25⁺ regulatory T cells such that the activated CD4⁺CD25⁺ regulatory T cells are able to suppress CD4⁺ CD45RA+CD25−T cell proliferation in vitro in an allogenic mixed lymphocyte reaction in which the activated CD4⁺CD25⁺ regulatory T cells are cocultured with allogenic CD4⁺CD45RA+CD25−T cell in the presence of CD2-depleted peripheral blood mononuclear cells that are syngenic for the CD4⁺CD25⁺ regulatory T cells.
 2. A therapeutic composition comprising the humanized antibody according to claim
 1. 3. A method comprising: preparing a therapeutic composition comprising the humanized anti-CD4 antibody according to claim
 1. 